The present invention relates to efficient methods for purifying a protein complex and its individual components comprising the complex from a cell, a cell lysate or tissue lysate or an organism.
In living cells, complex processes are typically accomplished by highly specific binding interactions among functional cell components, most commonly involving one or more proteins. Understanding which proteins bind to one another, and under what circumstances, poses difficult unsolved problems. An approach to learning which proteins bind to each other to form protein complexes is to isolate functional protein complexes, or portions thereof, in order to identify their components.
Recent advances in human genomics research have led to rapid progress in the identification of novel genes. In biological and pharmaceutical research, there is a need to determine the functions of gene products. An important step in defining the function of a novel gene is to determine its interactions with other gene products in the appropriate context. Several approaches have been devised towards this goal of determining a novel gene product's physical interaction with other gene products.
Specifically, yeast two-hybrid systems have been extensively employed to define interactions existing among proteins. The principles and methods of the yeast two-hybrid system have been described in detail elsewhere [Bartel and Fields (1997) The Yeast Two-Hybrid System, Oxford University Press, New York; Fields and Song (1989) Nature 340:245-246]. However, the number of false-positives and false-negatives arising from yeast two hybrid screening is high and thus extensive experimentation is required to validate the interactions observed in this system. Moreover, a yeast two hybrid experiment results in the identification of a pair of directly interacting proteins. For this reason, yeast-two hybrid is not directly useful for identifying higher order structures in multiprotein complexes comprised of both direct and indirect protein associations.
There is a continuing need for the discovery of additional protein-protein interactions that are physiologically relevant. The present invention provides an efficient method for purifying protein complexes from a cell, cell lysate, tissue lysate or organism by employing a combined set of affinity tags attached to a known protein as bait. The invention further provides an efficient method for purifying individual components comprising the complex from the excess bait used in the protein complex purification process. The interacting proteins purified according to the invention are identified by employing various art-known methods including mass spectrometric analyses. The advantages of the invention will be evident in the following description.